LOCUS       pCMV-EGFP-PHR(E2        6671 bp    DNA     circular SYN 22-OCT-2025
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6671)
  AUTHORS   Institute for Basic Science
  TITLE     Direct Submission
  JOURNAL   Exported Oct 22, 2025 from SnapGene 8.1.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6671
                     /mol_type="other DNA"
                     /note="color: #ffffff"
                     /organism="recombinant plasmid"
     promoter        1..591
                     /label=CMV
                     /note="color: #ffffff; direction: RIGHT"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #ffffff"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /note="color: #ffffff; direction: RIGHT"
     CDS             join(603..605,606..608,609..1319)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /note="This feature has 3 segments:
                        1:  603 ..  605 / #05fd14
                        2:  606 ..  608 / #05fd14 / 1a
                        3:  609 .. 1319 / #05fd14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1329..2822
                     /codon_start=1
                     /label=CRY2(E281A)
                     /label=PHR'
                     /note="color: #33cccc"
                     /translation="MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQ
                     FYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLY
                     DPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSI
                     ESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLN
                     EFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGA
                     ESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPL
                     VDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQY
                     ISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKA
                     SGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAA"
     primer_bind     complement(2146..2177)
                     /label=STIM1(E281A)-R
                     /note="color: black; sequence: 
                     GCTTTCGGCGCCTTCGCTGTTTTTATCGCGAG"
     primer_bind     2163..2192
                     /label=PHR(E281A)-F
                     /note="color: black; sequence: 
                     GAAGGCGCCGAAAGCGCCGATCTGTTCCTG"
     CDS             2823..2849
                     /codon_start=1
                     /product="CRY2clust"
                     /label=9 amino acids
                     /note="color: #ffffff"
                     /translation="ARDPPDLDN"
     CDS             2928..3326
                     /codon_start=1
                     /label=STIM1(318-450)
                     /note="color: #ff0000"
                     /translation="EEELEQVREALRKAEKELESHSSWYAPEALQKWLQLTHEVEVQYY
                     NIKKQNAEKQLLVAKEGAEKIKKKRNTLFGTFHVAHSSSLDDVDHKILTAKQALSEVTA
                     ALRERLHRWQQIEILCGFQIVNNPGIHSL"
     polyA_signal    3459..3580
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     rep_origin      complement(3587..4042)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        4069..4173
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     promoter        4175..4532
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
                     /note="color: #ffffff; direction: RIGHT"
     rep_origin      4383..4518
                     /label=SV40 ori
                     /note="SV40 origin of replication"
                     /note="color: #ffff00"
     CDS             4567..5361
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /note="color: #ccffcc"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    5593..5640
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
                     /note="color: #a6acb3"
     rep_origin      5969..6557
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcca
      601 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
      661 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
      721 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
      781 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
      841 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
      901 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
      961 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1021 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1081 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1141 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1201 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     1261 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagc
     1321 caccggtcat gaagatggac aaaaagacca tcgtctggtt tcggagagat ttgagaatag
     1381 aagataatcc cgcgctcgcc gccgcggccc acgagggttc cgtcttcccc gttttcattt
     1441 ggtgtcctga agaagaaggc cagttttatc ccggaagggc ctctaggtgg tggatgaagc
     1501 aaagtctggc ccatcttagc cagtcactga aagcactggg cagtgatctt accctgatca
     1561 agacacacaa taccatctct gccatcctcg actgcatcag ggtgaccggc gcaacgaaag
     1621 tcgtgtttaa ccacctgtac gatccagtta gtctggtgcg cgaccacact gtgaaggaga
     1681 agctggtgga acgggggatc agtgtgcaga gctacaacgg ggaccttctg tacgagccat
     1741 gggagatcta ttgcgagaaa gggaaaccgt tcacctcctt caacagttac tggaagaaat
     1801 gtttggatat gtcaatagag tccgttatgt tgccccctcc ctggagactg atgccgatta
     1861 ctgctgctgc agaggccatc tgggcctgct ccatcgagga actcggtctg gaaaatgaag
     1921 cagaaaagcc aagcaatgca cttctcacta gagcctggag ccccggctgg tctaatgccg
     1981 acaagctgct taacgagttc atcgaaaaac aactgattga ctacgcgaag aactccaaga
     2041 aagtggtagg taactcaact agcttgctct ctccatatct ccattttggc gagatttctg
     2101 tccgccatgt atttcagtgc gctcggatga aacagattat ctgggctcgc gataaaaaca
     2161 gcgaaggcgc cgaaagcgcc gatctgttcc tgcgagggat cggacttcgg gaatactccc
     2221 ggtatatatg tttcaacttt ccattcacac acgagcagag tctgttgtcc cacctcaggt
     2281 tcttcccctg ggacgccgat gtcgacaaat tcaaggcatg gagacaggga aggacagggt
     2341 acccactcgt ggatgctggc atgagagagc tctgggctac aggctggatg cacaaccgca
     2401 tccgggtaat cgtgtcctca tttgctgtca agtttctgct cctgccttgg aaatggggaa
     2461 tgaagtactt ttgggatacc cttctcgacg ccgacttgga gtgtgacatt ctgggatggc
     2521 aatatattag cgggtcaatt cctgacggcc atgagttgga caggttggac aatccggcct
     2581 tgcagggagc taagtatgat cccgaaggag agtatattcg acagtggctc cccgagctgg
     2641 cccgacttcc tacggagtgg attcaccatc cttgggacgc accactgaca gtgctcaagg
     2701 caagcggggt ggagctgggc accaattacg ctaagcctat agttgatata gatacagcac
     2761 gcgagctgct ggctaaagcg atctctcgca ctcgggaggc gcagattatg atcggtgctg
     2821 ccgccaggga cccccccgac ctcgacaacg aattcagtgg gggaggcgga gggggtggtt
     2881 caggtggtgg tggtggttcg ggtggtggcg gagggggagg ctccggagag gaggagttgg
     2941 agcaggttcg ggaggccttg aggaaagcag agaaggagct agaatctcac agctcatggt
     3001 atgctccaga ggcccttcag aagtggctgc agctgacaca tgaggtggag gtgcaatatt
     3061 acaacatcaa gaagcaaaat gctgagaagc agctgctggt ggccaaggag ggggctgaga
     3121 agataaaaaa gaagagaaac acactctttg gcaccttcca cgtggcccac agctcttccc
     3181 tggatgatgt agatcataaa attctaacag ctaagcaagc actgagcgag gtgacagcag
     3241 cattgcggga gcgcctgcac cgctggcaac agatcgagat cctctgtggc ttccagattg
     3301 tcaacaaccc tggcatccac tcactgtagg gatccaccgg atctagataa ctgatcataa
     3361 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     3421 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     3481 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     3541 attctagttg tggtttgtcc aaactcatca atgtatctta acgcgtaaat tgtaagcgtt
     3601 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     3661 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     3721 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     3781 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     3841 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     3901 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     3961 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     4021 aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4081 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4141 taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga
     4201 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4261 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     4321 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     4381 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     4441 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     4501 gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg
     4561 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4621 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4681 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4741 gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4801 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4861 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4921 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4981 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     5041 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg
     5101 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     5161 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     5221 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     5281 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     5341 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     5401 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     5461 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5521 tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     5581 cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc
     5641 ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt
     5701 ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag
     5761 gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat
     5821 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5881 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5941 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6001 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6061 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     6121 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6181 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6241 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6301 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6361 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6421 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6481 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6541 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6601 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6661 ccgccatgca t
//
